Effects of Imipenem-containing Niosome nanoparticles against high prevalence methicillin-resistant Staphylococcus Epidermidis biofilm formed

We aim to assess the antibacterial and anti-biofilm properties of Niosome-encapsulated Imipenem. After isolating Staphylococcus epidermidis isolates and determining their microbial sensitivity, their ability to form biofilms was examined using plate microtiter assay. Various formulations of Niosome-encapsulated Imipenem were prepared using the thin-film hydration method, Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Inhibitory Concentration (MIC) were determined, and biofilm genes expression was examined. Drug formulations’ toxicity effect on HDF cells were determined using MTT assay. Out of the 162 separated S. epidermidis, 106 were resistant to methicillin. 87 MRSE isolates were vancomycin-resistant, all of which could form biofilms. The F1 formulation of niosomal Imipenem with a size of 192.3 ± 5.84 and an encapsulation index of 79.36 ± 1.14 was detected, which prevented biofilm growth with a BGI index of 69% and reduced icaD, FnbA, EbpS biofilms’ expression with P ≤ 0.001 in addition to reducing MBIC and MIC by 4–6 times. Interestingly, F1 formulation of niosomal Imipenem indicated cell viability over 90% at all tested concentrations. The results of the present study indicate that Niosome-encapsulated Imipenem reduces the resistance of MRSE to antibiotics in addition to increasing its anti-biofilm and antibiotic activity, and could prove useful as a new strategy for drug delivery.

www.nature.com/scientificreports/ transferase) makes up a major part of the exopolysaccharide matrix of the biofilm 6 . Biofilm strength and maturity are then facilitated due to the expression of the FnbA and EbpS elastin genes 7 . FnbA contains multiple fibronectin (Fn) substituents and binding regions, each capable of joining both immobilized and soluble forms of Fn. This enables S. epidermidis to attack endothelial cells in vivo and in vitro with no need for additional factors 8 . The elastin-binding protein (EbpS) is a 25 kDa cell surface protein encoded by the EbpS gene. The binding of S. epidermidis to the 30 kDa N-terminal region of elastin, which is the main component of elastic fiber extracellular matrix, leads S. epidermidis to colonize the tissue 9 . The most important antibiotics selected for the experimental treatment of infections from S. epidermidis (MRSE) methicillin-resistant strains are imipenem and vancomycin. Imipenem is a broad-spectrum antibiotic used to treat various types of bacterial infections, including pneumonia, meningitis, sepsis, and anthrax. Imipenem is usually prescribed as a strategic drug to treat patients suffering from systemic infections. According to previous research, using imipenem together with other antibiotics increases their strength against MRSE strains [5][6][7][8][9] . However, imipenem overuse over the last decade has resulted in an increased population of enterococci with the imipenem resistance gene. Hence, there is a high probability of plasmids containing resistance genes transferring to S. epidermidis strains through conjugation 10 . The bacterium's resistance to antibiotics is due in part to its biofilm structure. This bacterium's methicillin-resistant forms have recently sparked widespread alarm. Because of the increasing frequency of methicillin resistance, hazardous antibiotics such the glycol-peptide vancomycin are now used to treat gram-positive infections. However, intermediate vancomycin resistance was discovered in two strains of S. epidermidis and S. hemolyticus. This is concerning since there are currently few viable choices for treating staphylococcal infections [11][12][13] . Researchers are using nanotechnology to produce more novel antibiotics as a result of the rise of bacterial resistance and indeed the slow speed of establishing novel antibiotics in recent decades. Nanomaterials have become one of the best choices for bacterial strain suppression due to their low cost of manufacturing and lack of environmental impact 14 . Encapsulation of antimicrobial agents in nanocarrier systems is one of the most promising and effective approaches to increase antibacterial activity while minimizing adverse effects 15,16 . Niosomes have recently gained popularity as a means of improving selective medication delivery and the therapeutic effectiveness of antifungal agents. Niosomes are bilayer structures with nonionic surfactants that make them water soluble and allow them to transport large amounts of medication 17 . Niosomes contain unique properties that might be utilized to encapsulate a variety of medicines 18 . The usage of Niosomes as antimicrobial nanocarriers has recently piqued researchers' interest 19 . As a result, imipenem was chosen as a powerful antibiotic against MRSE strains in the current investigation to minimize antibiotic resistance and boost antimicrobial effects. The goal of this research was to create Niosome-encapsulated imipenem with enhanced antibacterial activity against MRSE strains.

Materials and methods
Ethics approval and consent to participate.  (Table 1). After adding glass beads to all 2 compounds, drug compound solvents were evaporated using a rotary evaporator (Heidolph, Germany) for one hour at 60 °C and 120 rpm rotation. Afterward, dried thin films were hydrated for one hour using a solution of imipenem dissolved in 10 ml of PBS at 60 °C with a speed of 120 rpm to obtain www.nature.com/scientificreports/ different Niosome formulations. The minimum inhibitory concentration of imipenem against Staphylococcus (1 mg/ml) was used as the concentration of this drug for encapsulation. The resulting particles were sonicated using a probe sonicator in an ice bath using SONOPULS ultrasonic homogenizers (amplitude: 25%, 200 wt) for five minutes to reduce the size of Niosomes containing imipenem, and samples were stored at 4 °C for subsequent experiments.
Noisome-encapsulated imipenem morphological characteristics. The average size, polydispersity index, and zeta potential of imipenem loaded in noisome were obtained using dynamic light scattering (DLS) and ZetaPlas palladium electrodes (Brookhaven Instruments Corp., USA). The polydispersity index (PDI) is a size-based metric for assessing sample variability. Polydispersity can develop as a result of a pattern's particles size or accumulation or aggregation during extraction or evaluation. PDI was measured by DLS (Brookhaven Instruments Corp., USA). Various newly prepared Niosome formulations were diluted two times using distilled water at a ratio of 20:1 to prevent multiple scattering as a result of interactions between particles, and analysis was conducted at 25 °C with a 90-degree light scattering angle. The average z diameter and Niosome multiple scattering index were determined, and their zeta potentials were measured. Niosome-encapsulated imipenem particles were coated with a gold layer to generate electrical conductivity and were examined using an electron microscope (SEM) device model MIRA3 (TESCAN, Czech Republic).
Entrapment efficiency (EE). Encapsulation efficiency was obtained by determining the amount of noncapsulate imipenem (free imipenem) in the formed Niosomes. Noisome-encapsulated imipenem particles were isolated over an hour at 4 °C in a refrigerated centrifuge at 1400 rpm. The imipenem content of the supernatant was examined through ELISA Reader Stat Fax2100's (Awareness Technology, Ukraine) light absorbance reading of the supernatant at 276 nm wavelength, and the EE percentage was obtained based on Formula (1).
Evaluation of noisome-encapsulated release and stability. Dialysis was employed to examine imipenem's release of the noisome. The dialysis tube was soaked in distilled water for 24 h. Then, 0.5 ml (10 mg) of imipenemloaded noisome was placed in a dialysis bag, and 0.5 ml imipenem antibiotic aqueous solution containing 10 mg imipenem was also used as a control sample. Dialysis bags were immersed in conical flasks in 75 ml distilled water and shaken at 50 rpm in a water bath at 37 °C. Five milliliters were withdrawn from the receptor medium at intervals of one, two, four, six, 12, and 24 h, Aliquots of samples were replaced with a new medium at 37 °C, and imipenem was measured by spectrophotometry at 281 nm. The diffusion profile was determined using various kinetic models. This method was used to monitor the stability of diffusion of various Niosome-encapsulated imipenem formulations at intervals of 7, 14, 21, 28, 35, 42, 49, and 56 days for a storage period of two months at 25 °C.
Investigation of antibacterial activity. Bacterial isolation and antibiotic susceptibility. This descriptive study collected a total of 300 clinical samples from Shariati (150 samples), Firoozgar (150 samples), in Tehran. The samples used are from hospitals bio-bank. Samples were cultivated in blood agar cultures using sterile swabs at 37 °C for 24 h. First, S. epidermidis isolates were detected using Gram staining, catalase, mannitol fermentation, and DNase tests. Then identification of the species was performed by analyzing the 16S rRNA gene. Strain sensitivity to various antibiotics was examined by disc diffusion according to the Clinical and Laboratory Standards Institute (CLSI, CLSI supplement M100 (ISBN 1-56238-804-5 (Print); ISBN 1-56238-805-3 (Electronic)) method after S. epidermidis strains were detected and confirmed. S. epidermidis isolates' sensitivity to vancomycin (VA, 10 μg), cefoxitin (FOX, 10 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CD, 2 μg), mupirocin (MU, 5 μg) and imipenem (IMI, 10 μg) (MAST, UK) discs was measured in Muller Hinton agar culture (Merck, Germany). It must be noted that cefoxitin antibiotic discs were used to detect MRSE methicillin resistance, and the S. epidermidis ATCC 14990 standard strain was used as the positive control. Multidrug-resistant (MDR) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.
DNA extraction process. The bacterial genomic DNA was extracted according to the directions of the Cinna Gene company's extraction kit (Cinna Pure DNA KIT, Alborz, Iran). Briefly, the bacterial solution was centrifuged. After centrifuging the bacterium, the precipitate was mixed with 100 μl of proteinase K and protease buffer and heated at 55 °C for 30 min. Lysis buffer was introduced in 200 μl and vortexed for 15 s. Afterward, 300 μl of precipitation solution was added and inverted ten times before being subjected to -20 °C for one hour.
The supernatant was drained after 10-min centrifugation at 13,000 rpm, then 700 μl of wash buffer was added and centrifuged for 5 min at 13,000 rpm. The PCR test was used to identify 16S rRNA gene. www.nature.com/scientificreports/ µL icaD reverse primer, 0.5 µL icaD forward primer, 0.5 µL FnbA reverse primer, 0.5 µL FnbA forward primer, 0.5 µL EbpS reverse primer, 0.5 µL EbpS forward primer, 2 µL template DNA, and 15 µL distilled water (Amplicon, Denmark). The PCR program was performed in the form of an initial denaturation at 95 °C for five minutes and 35 initial denaturation cycles for one minute at 94 °C, one minute of annealing at 58 °C, one minute of extension at 72 °C, and a final extension at 72 °C for five minutes. The amplified products were investigated through 1% agarose gel electrophoresis to detect the desired genes of vancomycin resistance. Enterococcus faecalis ATCC 51299 were used as Vancomycin-resistant control.

Molecular detection of icaD, FnbA, and EbpS biofilm genes and resistance to vancomycin
Antibacterial activity of noisome-encapsulated imipenem. The antibacterial activity of various noisome-encapsulated imipenem formulations against MRSE strains was examined through broth microdilution. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were then determined as follows. The MIC is the lowest concentration of a chemical, usually a drug, which prevents visible growth of a bacterium. The MBC is measured by sub-culturing the broths used for MIC determination onto fresh agar plates. MBC is the lowest concentration of a drug that results in killing 99.9% of the bacteria being tested. To determine the MBC, the dilution representing the MIC and at least two of the more concentrated test product dilutions are plated and enumerated to determine viable CFU/ml. The closer the MIC is to the MBC, the more bactericidal the compound. After culturing S. epidermidis in Müller-Hinton broth for 24 h, 5 × 10 5 CFU/mL (5 μl) bacteria, 100 μl of various Niosome-encapsulated imipenem formulations, free imipenem and free Niosome (at a concentration ranging from 0.03 to 64 μg/ml), and 95 μl pure Hinton broth were poured into each well of a 96-well plate and incubated for 24 h at 37 °C. The sub-MIC values of negative control well (containing pure culture medium), and positive control well (containing culture medium plus standard S. epidermidis strain ATCC 14,990) were determined, and the test was replicated for a 3 time.
Inhibition of biofilm formation. Sub-MIC concentrations of free imipenem and Niosomes and various formulations of Niosome-encapsulated imipenem were used to determine the minimum biofilm inhibition concentration (MBIC) against the S. epidermidis strains. The wells of the 96-well plate were filled with 100 μl drug sample and 100 μl of the bacteria cultured in Müller Hinton Broth. The plates were incubated for 48 h at 37 °C and stained with crystal violet 1% after being washed. The tests were replicated two times, and the mean MBIC was determined to be OD630 < 0.1.
Analysis of biofilm gene expression. The expression of the biofilm genes icaD, FnbA, and EbpS was examined through polymerase chain reaction (qRT-PCR) using the specific primers indicated in Cytotoxicity study. According to standard ISO10993-5 and Li et al. 22 , the cytotoxicity test was performed by the MTT method on HDF normal cells. To check the cytotoxicity, an L929 confluence flask was placed under a hood, and the cells were cultured in fully sterile DMEM with high Glu (catalog number 11965118) (Thermo Fisher Scientific, United States) culture medium 23 . The surface of the cells was washed with PBS, and trypsin was added to remove the cells from the fibroblastic state and make them spherical. Then, DMEM containing 10% FBS (bovine fetal serum) was poured onto the cells to neutralize trypsin, and the cells were poured into a Falcon tube and centrifuged at 1200 rpm for 5 min. The supernatant was discarded, DMEM containing 10% FBS was poured, and cell counts were performed on a Neobar slide. For each well, a 24-cell plate of 5 × 10 4 cells were con- www.nature.com/scientificreports/ sidered. Sterile samples of free imipenem and niosomal encapsulated imipenem were also placed in the center of each plate under sterile conditions, and the cell suspension was added and placed in a 37 °C incubator containing 5% CO 2 for 24 h. According to standard ISO10993-5, after 24 h, the medium was removed, the cells were washed with PBS, and then a sufficient amount of MTT (1 mg/ml) was poured onto the cell layer. The culture plate was incubated for 3 to 5 h, and the cells were then washed with PBS to remove unreacted MTT. The product of Formosan solvent by isopropanol and OD560 nm of each cell was evaluated. Cell viability was obtained from the absorption fraction of each sample on the control sample (polystyrene container). Additionally, the percentage of living cells or the rate of cell survival was calculated based on the following Eq. (2)

Results
Imipenem encapsulated in Niosomes has a uniform and spherical structure with suitable morphological features. Various formulations of Niosome-encapsulated imipenem were studied morphologically and in terms of various indicators, such as the surfactant-to-cholesterol ratio, Span-to-Tween ratio, and the composition of different surfactants Span and Tween ( Table 1). Each of the formulations had a distinct size, polydispersity index (PDI), and entrapment efficiency (EE). Dynamic light scattering (DLS) revealed that various formulations of Niosome-encapsulated imipenem had good uniformity ( Table 3). As demonstrated, F1 is of a smaller and better size and is associated with surfactant Span60's hydrophile-lipophile balance. Span60 has a hydrophile-lipophile balance of 4.7, while the corresponding value in Span40 is 6.7. Therefore, nanoparticles formulated with Span60 are smaller. In addition, the EE content of the F1 formulation was higher than that of the other formulations, which might be due to the surfactant used. A longer saturated alkyl chain is directly associated with the permeability of drugs in Niosomes so that longer saturated alkyl chains will result in greater permeability. Span60 has a longer alkyl chain than Span40, which is why formulations incorporating Span60 have higher indexes and EE content is at its peak in the F1 formulation. Polydispersity indexes (PDI) smaller than 0.3 indicate a suitable distribution of small nanoparticles, indicating that the F1 formulation is the optimal formulation given that it has the smallest PDI (Table 3). As Fig. 1 demonstrates, SEM results indicated a uniform spherical shape in the Niosome-encapsulated imipenem of the F1 formulation with an average size of 192.3, which indicates the suitable diameter (< 200) of this drug formulation.
The amount of imipenem released from the Niosome was examined using the dialysis bag method. Imipenem's antibiotic release from Niosome nanoparticles demonstrated a two-phase release pattern made up of the blast and stable phases so that the stable constant reached its peak after 7 h in various formulations. The released amount of antibiotic then started declining, and the release speed increased with a gentle slope for up to 72 h. Lower speeds in drug release in the blast phase were associated with higher encapsulation efficiency and more effective controlled drug release. Therefore, the highest efficiencies of controlled emission were attributed to formulations F1 and F2 with respective release percentages of 45 and 56 in the blast phase. The F1 formulation had the highest efficiency in controlled diffusion, showing 98.2% drug release after 312 h, while free imipenem drug release was 89% in the blast phase, which increased to 99% in 72 h ( Fig. 2A).
The morphological features of Niosome-encapsulated imipenem, such as the average size and encapsulation efficiency (EE), were examined to evaluate its stability at various temperatures and time intervals. The results showed that increasing the temperature affects the morphological aspects of Niosome-encapsulated imipenem and increases the size, resulting in a decrease in drug EE, but extending the life span had no significant influence on its EE or morphological features. (Fig. 2B,C). According to the results, various drug formulations had higher stability at 4 °C than at 25 °C, indicating the negative impact of temperature on encapsulated drug stability. On the other hand, the F1 formulation of Niosome-encapsulated imipenem was the most stable among the formulas.
The high frequency of MRSE strains is found in Iran Hospitals. Out of the 300 collected samples, 162 cases were confirmed to contain S. epidermidis after microbial diagnostic tests, and further molecular exami-(2) Viable cell percentage% = treated cells mean absorbance/control cells mean absorbance×100

Imipenem encapsulated in Niosomes has strong antibacterial and antibiofilm properties.
Two drugs of free imipenem and various compositions of Niosome-encapsulated imipenem were evaluated against MDR S. epidermidis (that were resistant to vancomycin and methicillin) to analyze the antimicrobial features of MIC, MBC, and sub-MIC Niosome-encapsulated imipenem, and the results are listed in Table 5. MIC values for free imipenem range between 4 and 8 μg/ml, to which the aforementioned strains indicated various degrees of resistance. In addition, MBC values did not indicate significant differences from MIC values. The antibiofilm activity of Niosome-encapsulated imipenem compared to free imipenem was also examined through MBIC. However, among all the examined samples, the F1 formulation decreased the amount of Niosome-encapsulated imipenem by 4-6 times the MBIC in comparison with free imipenem. Other formulations of Niosome-encapsulated imipenem (F2) indicated other MIC, MBC, and MBIC degrees. On the other hand, free imipenem had no lethal effect or growth inhibition at any of the aforementioned concentrations. Out of the examined bacterial strains, the 4 isolates SMRSE1, SMRSE2, SMRSE3, and SMRSE4 were selected as biofilm-generating strains, and the ability of Niosomes carrying imipenem to inhibit the growth of resistant S. epidermidis isolates over a short period of exposure to drug formulations was compared to that of free imipenem. Compared to the minimum biofilm inhibitory concentration (MBIC) study, biofilm growth inhibition conditions were harder since biofilms were first treated for only two hours with drug formulations or free imipenem; biofilms were then washed and incubated in antibiotic-free medium for 24 h. This study was conducted at concentrations ranging from 1/2 MIC to 2MIC for the respective isolates. The concentration of drug formulations compared to free imipenem MIC was calculated for physical composition, and the results were reported as the biofilm growth inhibition percentage (BIG%, Fig. 3A).
Afterward, subMIC concentrations of Niosome-encapsulated imipenem were used to examine antibiofilm activity, and the expression of biofilm genes icaD, FnbA, and EbpS in bacteria was examined through    (Fig. 4).

Discussion
Methicillin-resistant S. epidermidis is a prevalent hospital pathogen whose prevalence has increased worldwide over the past three decades and has posed serious threats as a nosocomial infection 24 . Hence, examining this pathogen's prevalence in samples taken from 2 hospitals, Shariati, Firouzgar, indicated a 54% S. epidermidis prevalence. On the other hand, S. epidermidis (MRSE) bacteria demonstrate resistance to a variety of antibiotics, and some of its strains have indicated vancomycin resistance 25,26 . The present study examined the sensitivity of various isolates, indicating that 82.71 of the isolates were MDR, which was consistent with previous research 27 .
In addition, 72.38% of the isolates were capable of forming biofilms, which are among the main factors of microbial resistance among Iranian nosocomial infections according to Piri et al. 28 . According to the literature, the formation of biofilms in S. epidermidis reduces the speed of antibiotic penetration and causes complications for the treatment of infections due to these bacteria 29 . Therefore, research focused on new treatment strategies as well as strategies for reducing MRSE resistance is of great importance. Given that Niosome formulations are being studied as promising tools to increase compounds' antibacterial activities 30 , the present study focused on Niosome-encapsulated imipenem to reduce the resistance of MRSE and increase antibacterial activity against S. epidermidis. Various synthesized formulations were prepared, including Tween 60 and Tween 40 nonionic surfactants as well as Span 60 and Span 40 mixed with cholesterol. Given that various cholesterol levels could result in various bilayers in formulations and impact bilayers' surface load, its amount was kept steady across all formulations. Therefore, the Niosome membrane's strength and resistance to ultrasound waves were kept constant across all formulations by keeping the cholesterol constant, which resulted in more uniform vesicles in terms of size 31 . On the other hand, various surfactant combinations resulted in nanostructures with various morphological features 32 . The use of equal Tween and Span ratios resulted in different results. Tween 60 results in reduced Niosome membrane strength because of its high hydrophilicity 33 . This defect was resolved by adding an equal Span 60 surfactant (50:50), which is more hydrophobic and results in the formation of dense Niosome films. On the other hand, Tween 60 and Span 60 have higher phase transfer temperatures than the other surfactants used in the study, which results in increased drug encapsulation 34 . Span60's long saturated alkyl chain resulted in more preamble Niosomes as an influential factor, resulting in multiplied drug encapsulation 35 . In addition, the Span60 surfactant has a lipophilic balance of 4.7, which is lower than that of Span40 (6.7), resulting in the formation of smaller vesicles 36 . Therefore, the F1 formulation was revealed to have a more suitable size and morphological features than the F2 formulations. The Span40 used in F2 formulations resulted in larger vesicles compared to that of F1 due to their high hydrophilic-lipophilic balance. On the other hand, formulation F2 contained fewer encapsulated drugs despite their larger size due to Span40's unsaturated alkyl chain, resulting in their lower encapsulation efficiency (EE). In addition, Formulation F2 had lower phase transfer temperatures and weaker wall strength due to the presence of Tween 60 and Tween 40 surfactants, which resulted in the formation of a thin structure and low encapsulation efficiency. Hence, the size obtained for the F1 formulation was quite smaller than the other formulations, indicating this formulation's higher EE. Hedayati et al. examined various Niosomeencapsulated drug formulations, reporting that the size of Niosome-encapsulated drugs varied across different formulations 37 . This study's-controlled drug release profile indicates a biphasic pattern. Given the Niosome bilayer structure, the drug might reside either in the center of the Niosome within the two layers or at the surface of the Niosome during encapsulation 38 . Therefore, the drugs on the surface start being released over the first hours of release, which results in the blast phase (fast drug release). After seven hours, surface drugs will have released, www.nature.com/scientificreports/ and encapsulated drugs residing at the center of the Niosome and on the bilayer membrane start being released, resulting in controlled drug release and the stable phase. On the other hand, the cholesterol present in the system stops the gel-to-lipid phase transfer in Niosome systems, which prevents the drug from leaking out of the Niosomes, resulting in controlled drug release over the long term and longer efficient drug release 39 . Because of its higher strength and good surface density, formulation F1 forms better links with cholesterol, resulting in a reduced drug delivery process. In addition, negative zeta potentials are due to the electrostatic repulsion between the particles according to previous research, resulting in higher Niosome stability 40 . Studying the zeta potential  www.nature.com/scientificreports/ and stability revealed that formulation F1 had the most stable Niosome-encapsulated imipenem. Additionally, the drug stability was higher at 4 °C than at 25 °C, which might be due to the lower Niosome bilayer mobility at 4 °C 41 . As a negative factor, the time of drug immobility has a direct relationship with nanoparticle size and an inverse relationship with the EE index. A longer drug immobility time results in a larger nanoparticle size due to particle accumulation or fusion, and the pressure resulting from nanoparticle accumulation on the Niosome bilayer might result in the diffusion of the layers and a reduction in their EE 42 . The present study mainly aimed to improve the antibacterial features of Niosome-encapsulated imipenem. Niosome-encapsulated imipenem had a much lower MIC than free imipenem, indicating the drug's increased efficiency and antibacterial activity and bacteria's lower drug resistance. Barakat 45 . Consistent with that of Machado and Akbari, the results of the present study indicate increased antibacterial activity in nanomaterial-encapsulated antibiotics 46,47 . According to Gupta et al., there are various absorption and fusion mechanisms between the Niosome and the bacterial wall that could result in increased targeted drug delivery 48 . The other aim of the present study was to examine Niosome-encapsulated imipenem's antibiofilm features in the form of biofilm formation inhibition, biofilm growth inhibition, and prevention or reduction of biofilm-involved gene expression. Niosome-encapsulated imipenem's MBIC was four to six times free imipenem in all the studied isolates, indicating the higher efficiency of nanoparticles compared to free drug. On the other hand, the F1 formulation of Niosome-encapsulated imipenem indicated the highest biofilm formation and growth inhibition. A variety of studies have suggested a direct relationship between the reduced efficiency of antibacterial materials and the ability to form biofilms 49 40 . Hence, the present study examined the impact of Niosome-encapsulated imipenem sub-MIC concentrations on icaD, FnbA, EbpS, and biofilm genes, revealing a considerable decline in the expression of these genes compared to free imipenem. In addition, the F1 formulation had the highest impact on reducing gene expression and was therefore identified as the optimal formulation. Reducing the expression of the aforementioned genes could result in the inhibition of transcription through a direct impact and cause a reaction between Niosome-encapsulated imipenem and transcription factors, which will result in the inhibition or reduction of the expression of such genes. These results are consistent with those of Lawson et al. (2010), revealing the impact of nanoencapsulation on increasing the inhibition of biofilms and reducing biofilm gene expression 51 . Abdelazizi et al. suggested that Niosome encapsulation of norfloxacin improves its antibiofilm properties and MDR bacterial biofilm formation 52 . The increased microbial resistance to various antibiotics has highlighted the need to find new antibacterial compounds that are nontoxic to mammalian cells 53 . The present study examined the toxicity of various Niosome-encapsulated imipenem formulations to HDFs through the standard method of MTT, indicating a higher cell viability rate in groups treated with Niosome-encapsulated imipenem than in groups treated with free imipenem. Formulation F1 of Niosome-encapsulated imipenem indicated the lowest cytotoxicity compared to other formulations, with a cell viability of over 90% over 24 h. The cytotoxicity of these formulations www.nature.com/scientificreports/ was due to the use of various Span:Tween surfactant ratios with high biodegradability, making these formulations suitable candidates for encapsulating imipenem as well as other drugs 54 .

Conclusions
Niosome-encapsulated imipenem is a new approach for restoring imipenem characteristics at a low cost, giving imipenem distinct new properties, such as increased targeted drug delivery, maintaining stability, and controlling drug release. According to this study, this drug formulation is more effective than free imipenem in treating infections due to more resistant Staphylococcus Epidermidis isolates that are resistant to a variety of antibiotics, especially vancomycin and methicillin, and the present study is the first to report the impacts of encapsulating imipenem in Niosomes on biofilm formation and growth inhibition, biofilm eradication, and distinct antimicrobial activity against MRSE clinical isolates. As a general result, nisosms are promising new drug systems for increasing the antibacterial effects of drugs whose antibacterial features depend on their formulation and composition. According to the present study's results, it could be inferred that Niosome encapsulation of imipenem increases its antibacterial and antibiofilm activities against MDR and MRSE S. epidermidis strains, and these formulations could be considered a new strategy for targeted drug delivery.

Data availability
The data that support the findings of this study are available upon reasonable request from the authors.